Observations that GnRH and its agonists directly inhibit gonadal function via binding to receptors on Leydig cells had prompted speculation that GnRH like factors are produced locally in the testis and serve as messengers between Sertoli and Leydig cells. We have recently reported partial isolation of at least two GnRH-like factors with estimated molecular weights of 6,000 and 68,000 from ethanol:chloroform:acetic acid extracts of rat by immunoaffinity and reverse phase high pressure liquid chromatography. The aim of this project is 1) to further characterize and purify these factors to homogeneity; 2) to localize their site of origin; and 3) to assess their biologic function. The assay systems to be used to quantify these factors include pituitary and Leydig cell radioreceptor assays, radioimmunoassay using an antibody raised against D-lys6GnRH, pituitary cell monolayer culture and mixed Sertoli cell-Leydig cell culture. The 51Cr-labelling will be used as an adjunct in bioassays to differentiate nonspecific toxic effects from inhibitory effects. Two different schemes of purification are proposed based on preliminary data on the biochemical nature of these two factors. Proposed scheme for purification of the smaller peptide includes ethanol:chloroform:acetic acid extraction, defatting, steroid removal, a preliminary extraction on disposable ODS cartridges followed by preparative reverse phase chromatography on a C18 column using trifluoroacetic acid:acetonitrile solvent system. Scheme for the larger protein involves acetic acid extraction, defatting, steroid removal, desalting, salt fractionation, removal of albumin by blue sepharose, preparative electrofocussing and preparative ion exchange chromatography using FPLC (Pharmacia). Histochemical staining and quantitation of bioactivity in different testicular compartments may help localize the intratesticular site of origin of this material. Experiments proposed to assess physiologic control include hypophysectomy, selective hormone replacement after hypophysectomy, unilateral and bilateral cryptorchidism, unilateral and bilateral efferent duct ligation and fetal irradiation.